Amylase, Alpha Assay

Method: That of (1951) wherein the reducing groups released from starch are measured by the reduction of 3,5-dinitrosalicylic acid. One unit releases from soluble starch one micromole of reducing groups (calculated as maltose) per minute at 25°C and pH 6.9 under the specified conditions.

Dilute to a concentration of 1-10 micrograms/ml. A minimum of three different concentrations in this range should be run.

Procedure

Adjust spectrophotometer to 540 nm and 25°C.

Using the maltose stock solution prepare a maltose standard curve as follows: In numbered tubes, prepare 10 maltose dilutions ranging from 0.3 to 5.0 micromoles per ml. Include two blank tubes with reagent grade water only. Into a series of corresponding numbered tubes pipette 1 ml of each dilution of maltose. Add 1 ml of dinitrosalicylic acid color reagent. Incubate in boiling water bath for 5 minutes and cool to room temperature. Add 10 ml distilled water to each tube and mix well. Read A540 versus micromoles maltose.